The present invention relates generally to the separation of biological samples in a fluid flow introduced into a separation chamber in the presence of an applied field. More particularly, the present invention presents an apparatus and method of continuous flow zone electrophoresis for separation of particulate materials or biological samples, including cells and sub-cellular components, in a separation chamber configured to enhance separation of the sample components. The foregoing is accomplished by amplifying peak-to-peak distance between sample components in a non-equilibrium continuous flow zone electrophoretic fractionator by selective manipulation of particle residence time. The fractionator is provided with an axial fraction collector and a positive gradient along the applied field in particle residence time and/or particle deflection rate.
In the approximately thirty years since its inception, continuous flow electrophoresis has emerged as a powerful method for separating cells and subcellular components. Because it is carrier free and gentle, continuous flow electrophoresis offers a fast and efficient alternative to centrifugation. In fact, since cellular function is more closely associated with surface charge than with volume or density, properties upon which centrifugation effectuates separation, continuous flow electrophoresis has greater potential for purifying functionally homogeneous cell populations. Moreover, because functional activity is substantially preserved and samples are efficiently recovered, continuous flow electrophoresis is particularly attractive for large scale purification.
Barriolier, V. J., et al, Z. Naturforsch, 136:754 (1958) were the first group to reduce a continuous flow electrophoretic fractionator to purify biomolecules. The technique was further refined by Hannig, K., Z. Anal. Chem. 181:244-254 (1961) and Strickler, A., et al, Annals N.Y. Acad. of Sci., 209:497-514 (1973). Early applications of continuous flow electrophoresis were restricted to the separation of soluble components such as proteins and other biomolecules; the technology was extended to cells and subcellular components. Hjerten, S., Cell Separation Methods, Amsterdam, The Netherlands: Elsevier/North-Holland Biomedical Press, B.V., Bloemendal, H., ed., 127 (1977).
The principal of continuous flow electrophoresis entails establishing a conveying buffer flow in a first direction, either upwards or downwards, relative to gravity, in a narrow gap between two flat parallel plates. A sample is injected into the buffer at the center of the gap as a stream. The sample typically contains charged sub-populations A and B. The elements of A have a greater surface charge density (.sigma.), and, therefore, a greater mobility (.mu.), than those of B. Thus, .sigma..sub.A &gt;.sigma..sub.B, and .mu..sub.A &gt;.mu..sub.B. As the elements of A and B are conveyed by the buffer along an axial direction Z, they migrate towards the oppositely charged electrode, i.e., negatively charged components will migrate toward the anode. Since .mu..sub.A &gt;.mu..sub.B, the elements of A migrate faster than the elements of B. Consequently, by the time the sample reaches the end of the chamber, the elements of A and B are separated by some peak to peak distance .delta..sub.AB. The separated sample components are collected by a fraction collector.
An inherent limitation of continuous flow electrophoresis in the currently available apparatus and methods, is that the peak to peak distance (.delta..sub.AB) is often too small to fractionate the sample into substantially biologically pure components. It has been found that this is due to i) the sub-populations of most biological samples differ minutely in charge density; ii) the fraction collector exit receptacle must have an internal diameter of at least 0.5 mm to avoid obstruction and non-uniform flow; iii) the deflection of the sample towards the anode or cathode, which determines .delta..sub.AB, must be on the order of a 1 cm separation to maintain stable flow; and iv) the finite size of the injection tube and the dispersion of the sample during electrophoresis typically lead to overlapping peaks.
The inventors have found that with the current state of continuous flow electrophoresis, particles can be effectively fractionated only if their relative mobilities differ by at least 10%. The degree to which mobilities of the particles limit sensitivity of the continuous flow electrophoresis can be estimated by expressing the sensitivity (S) in terms of the smallest mobility interval .DELTA..mu.=(.mu..sub.A -.mu..sub.B) that can be fractionated into pure components A and B. Assuming that .delta..sub.AB must be at least twice the diameter of the receptacle, i.e., .delta..sub.AB must be greater than or equal to 2.DELTA.=1 mm, to isolate A from B without unacceptable overlap, then S may be expressed as: EQU S=(.mu..sub.A -.mu..sub.B).sub.min =(.DELTA..mu.).sub.min for which .delta.=1 mm
To evaluate the S, the relationship between .delta..sub.AB and .DELTA..mu./.mu. is derived. By definition of particle mobility .mu., the average net deflection (D) of the sample from the axis of introduction in a given Y-Z plane is related to the applied field strength (E) and the net residence-time (.tau.) of the sample in that plane, by the following equation: EQU D=.mu.E.tau.
where .mu.=(.mu..sub.A -.mu..sub.B)/2 is the average net mobility of the sample.
Similarly, the average net deflection of the two sample components D.sub.A and D.sub.B are: EQU D.sub.A =.mu..sub.A E.tau. EQU D.sub.B =.mu..sub.A E.tau.
Thus, the peak to peak distance between sample components .delta..sub.AB may be expressed as: EQU .delta..sub.AB =D.sub.A -D.sub.B =(.mu..sub.A -.mu..sub.B)E.tau.=.DELTA..mu.E.tau.
substituting for E .tau. gives: EQU .delta..sub.AB =(.DELTA..mu./.mu.)D, or .DELTA..mu./.mu.=.delta..sub.AB /D
substituting for .DELTA..mu./.mu. for .delta.=1 mm, we get: EQU s=(.DELTA..mu./.mu.).sub.min =1/D.sub.max
where D.sub.max is the maximum possible deflection of the sample components. It has been found that, under terrestrial conditions, D has to be on the order of 1 cm to maintain stable flow, assuming .mu. to be approximately 1 .mu.m/sec per V/cm of applied field. Thus, (.DELTA..mu./.mu.).sub.min must be on the order of 0.1 or 10%, an estimate which compares well with published values.
The 10% variance in relative component mobility can sometimes be overcome by selectively altering the surface charge of a sub-population via chemical modification of the surface groups. However, this approach requires that specific strategies must be developed on a case by case basis and may lead to changes in functional activity.
Resolution in continuous flow electrophoresis is also limited by wall effects such as electroosmosis and transverse gradients in sample residence time and temperature. The combination of these factors leads to the well-known crescent effect. While the crescent effect may be partially controlled by manipulating electroosmosis, this approach has limited applicability where the flow velocity profile deviates from the ideal parabolic transverse shape. Such deviations are more problematic under terrestrial conditions where buoyancy driven phenomena, such as thermal convection and particle and zone sedimentation are significant.
The problem of temperature variation is also important in scaling up continuous flow electrophoresis. It is known that a 17.degree. C. temperature differential exists between the wall and center of a 0.5 cm thick chamber, even where both walls of the chamber were cooled. Saville, D. A., PCH Physico Chemical Hydrodynamics, 1:299 (1980). Since particle mobility increases about 3% per degree centigrade (Hannig, K., et al, Biochemistry and Diagnostics, Git Verlag, GMBH, 93 (1990)) flow band broadening would result from such a temperature drop. Thermal convection also seriously limits scaling up of continuous flow electrophoresis. Efficient heat removal is not feasible if chamber thickness exceeds 1 mm.
Particle and zone sedimentation are two other buoyancy-induced phenomena that affect separation resolution and scale up potential. Particle sedimentation can lead to artifactual broadening of bands where particle size is on the order of 1 .mu.m or more, whereas zone sedimentation limits concentration of the sample stream.
Under microgravity, however, particle and zone sedimentation cease to be limiting. However, secondary field effects such as ohmic heating and electrohydrodynamics become limiting. Peeters, H., Cell Separation Methods, Amsterdam, The Netherlands: Elsevier/North-Holland Biomedical Press, B.V., Bloemendal, H., ed., 162 (1977). In this case, the lack of buoyancy induced convection and poor thermal conductivity of the buffer lead to sample overheating. Saville, supra, demonstrated that, under typical operating conditions, the gap width of a separation chamber cannot be increased over 0.75 cm without overheating the sample.
Ohmic heating poses a limitation on the ionic strength of the buffer since heat generated by the electric field increases with conductivity of the buffer, and therefore with the ionic strength of the buffer. The need to minimize ohmic heating limits the factors of sample concentration and residence time.
Electrohydrodynamics degrades resolution through deformation of the sample stream. Rhodes, P. H., et al, J. Colloid. Interface Science, 129(1):90 (1989). The degree of sample deformation varies with the ration of sample to buffer conductivity. Thus, sample concentration is directly limited and sample throughput is compromised.
Early attempts to amplify peak to peak distance were restricted to batch processes, such as those described by Biggin in 1983 or Ansorge in 1984, with buffer gradient and wedge shaped gels, respectively. The only known attempts at amplifying peak to peak distance in continuously operated fractionators has been achieved by selective manipulation of the surface-charge of the sub-populations through chemical modification of the surface groups.
The present invention achieves amplification of peak to peak distance by selectively manipulating particle residence-time and/or particle deflection rate. This approach has not heretofore been reported.